Breast cancer (BCa) is the most common cancer diagnosis in women and the second-leading cause of cancer-related death among women (Ries L A G, et al. (eds). SEER Cancer Statistics Review, 1975-2003, National Cancer Institute, Bethesda, Md.). Major advances in breast cancer treatment over the last 20 years have led to significant improvement in the rate of disease-free survival (DFS). For example, therapies utilizing antibodies reactive against tumor-related antigens have been used to block specific cellular processes in order to slow disease progress or prevent disease recurrence. Despite the recent advances in breast cancer treatment, a significant number of patients will ultimately die from recurrent disease.
Vaccines are an attractive model for preventing, slowing, or prohibiting the development of recurrent disease due to their ease of administration, and because of their high rate of success observed for infectious diseases. The basic concept of constructing a cancer vaccine is straightforward in theory. The development of effective cancer vaccines for solid tumors in practice, however, has met with limited success. For example, one group attempting to administer a peptide vaccine directed against metastatic melanoma observed an objective response rate of only 2.6% (Rosenberg S A et al. (2004) Nat. Med. 10:909-15).
There are many potential explanations for this low success rate (Campoli M et al. (2005) Cancer Treat. Res. 123:61-88). For example, even if an antigen is specifically associated with a particular type of tumor cell, the tumor cells may express only low levels of the antigen, or it may be located in a cryptic site or otherwise shielded from immune detection. In addition, tumors often change their antigenic profile by shedding antigens as they develop. Also contributing to the low success rate is the fact that tumor cells may express very low levels of MHC proteins and other co-stimulatory proteins necessary to generate an immune response.
Additional problems facing attempts at vaccination against tumors arise in patients with advanced-stage cancers. Such patients tend to have larger primary and metastatic tumors, and the cells on the interior of the tumor may not be accessible due to poor blood flow. This is consistent with the observation that vaccine strategies have tended to be more successful for the treatment of hematologic malignancies (Radford K J et al. (2005) Pathology 37:534-50; and, Molldrem J J (2006) Biol. Bone Marrow Transplant. 12:13-8). In addition, as tumors become metastatic, they may develop the ability to release immunosuppressive factors into their microenvironment (Campoli, 2005; and, Kortylewski M et al. (2005) Nature Med. 11:1314-21). Metastatic tumors have also been associated with a decrease in the number of peripheral blood lymphocytes, and dendritic cell dysfunction (Gillanders W E et al. (2006) Breast Diseases: A Year Book and Quarterly 17:26-8).
While some or all of these factors may contribute to the difficulty in developing an effective preventative or therapeutic vaccine, the major underlying challenge is that most tumor antigens are self antigens or have a high degree of homology with self antigens, and are thus expected to be subject to stringent immune tolerance. Thus, it is clear that many peptide-based cancer vaccines, with or without immune-stimulating adjuncts, may be doomed to only limited success in clinical practice due to low immunogenicity and lack of specificity.
Prototype breast cancer vaccines based on single antigens have been moderately successful in inducing a measurable immune response in animal experiments and in clinical tests with breast cancer patients. The observed immune response, however, has not translated into a clinically-significant protective immunity against recurrence of disease put in remission by standard therapy (e.g., surgery, radiation therapy, and chemotherapy).
HER2/neu is a proto-oncogene expressed in many epithelial malignancies (Slamon D J et al. (1989) Science 244:707-12). HER2/neu is a member of the epidermal growth factor receptor family and encodes a 185-kd tyrosine kinase receptor involved in regulating cell growth and proliferation. (Popescu N C et al. (1989) Genomics 4:362-366; Yarden Y et al. (2001) Nat Rev Mol Cell Bio 2:127-137.) Over-expression and/or amplification of HER2/neu is found in 25-30% of invasive breast cancers (BCa) and is associated with more aggressive tumors and a poorer clinical outcome. (Slamon D J et al. Science (1987) 235:177-182; Slamon D J et al. Science (1989) 244:707-12; Toikkanen S et al. J Clin Oncol (1992) 10:1044-1048; Pritchard K I et al. (2006) N. Engl. J. Med. 354:2103-11.)
Determining HER2/neu status is performed predominately via two tests, immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). IHC detects over-expression of HER2/neu protein and is reported on a semi-quantitative scale of 0 to 3+(0=negative, 1+=low expression, 2+=intermediate, and 3+=over-expression). FISH on the other hand detects amplification (excess copies) of the HER2/neu gene and is expressed as a ratio of HER2/neu gene copies to chromosome 17 gene copies and interpreted as “over-expression” if FISH is ≧2.0 copies. (Hicks D G et al. Hum Pathol (2005) 36:250-261.) Concurrence rate of IHC and FISH is approximately 90%. (Jacobs et al. J Clin Oncol (1999) 17:1533-1541.) FISH is considered the gold standard, as retrospective analysis reveals it is a better predictor of trastuzumab (Tz) response; it is more objective and reproducible. (Press M F et al. J Clin Oncol (2002) 14:3095-3105; Bartlett J et al. J Pathol (2003) 199:411-417; Wolff A C et al. J Clin Oncol (2007) 25:118-145.)
Identification and quantification of HER2/neu as a proto-oncogene has led to humoral or antibody-based passive immunotherapy, including the use of trastuzumab (Herceptin® Genentech Inc., South San Francisco, Calif.). Trastuzumab is a recombinant, humanized monoclonal antibody that binds the extracellular juxtamembrane domain of HER2/neu protein. (Plosker G L et al. Drugs (2006) 66:449-475.) Tz is indicated for HER2/neu over-expressing (IHC 3+ or FISH≧2.0) node-positive (NP) and metastatic BCa patients, (Vogel C L et al. J Clin Oncol (2002) 20:719-726; Piccart-Gebhart M J et al. N Engl J Med (2005) 353:1659-1672) and shows very limited activity in patients with low to intermediate HER2/neu expression. (Herceptin® (Trastuzumab), prescription product insert, Genentech Inc, South San Francisco, Calif.: revised September 2000.)
Another form of immunotherapy being pursued is vaccination and active immunotherapy targeting a cellular immune response to epitopes on tumor associated antigens, such as HER2/neu. HER2/neu is a source of several immunogenic peptides that can stimulate the immune system to recognize and kill HER2/neu-expressing cancer cells. (Fisk B et al. J Exp Med (1995) 181:2109-2117.) Two such peptides are termed E75 and GP2. E75 and GP2 are both nine amino-acid peptides that are human leukocyte antigen (HLA)-A2-restricted and stimulate CTL to recognize and lyse HER2/neu-expressing cancer cells (Fisk B et al. J Exp Med (1995) 181:2109-2117; Peoples G E et al. Proc Natl Acad Sci USA (1995) 92:432-436).
E75 is derived from the extracellular domain of the HER2/neu protein and corresponds to amino acids 369-377 (KIFGSLAFL)(SEQ ID NO:3) of the HER2/neu amino acid sequence and is disclosed as SEQ ID NO:11 in U.S. Pat. No. 6,514,942, which patent is hereby incorporated by reference in its entirety. The full length HER2/neu protein sequence is set forth below and is disclosed as SEQ ID NO:2 in U.S. Pat. No. 5,869,445, which patent is hereby incorporated by reference in its entirety:
(SEQ ID NO: 1)MKLRLPASPETHLDMLRHLYQGCQVVQGNLELTYLPTNASLSFLQDIQEV QGYVLIAHNQVRQVPLQRLRIVRGTQLFEDNYALAVLDNGDPLNNTTPVT GASPGGLRELQLRSLTEILKGGVLIQRNPQLCYQDTILWKDIFHKNNQLA LTLIDTNRSRACHPCSPMCKGSRCWGESSEDCQSLTRTVCAGGCARCKGP LPTDCCHEQCAAGCTGPKHSDCLACLHFNHSGICELHCPALVTYNTDTFE SMPNPEGRYTFGASCVTACPYNYLSTDVGSCTLVCPLHNQEVTAEDGTQR CEKCSKPCARVCYGLGMEHLREVRAVTSANIQEFAGCKKIFGSLAFLPES FDGDPASNTAPLQPEQLQVFETLEEITGYLYISAWPDSLPDLSVFQNLQV IRGRILHNGAYSLTLQGLGISWLGLRSLRELGSGLALIHHNTHLCFVHTV PWDQLFRNPHQALLHTANRPEDECVGEGLACHQLCARGHCWGPGPTQCVN CSQFLRGQECVEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGPE ADQCVACAHYKDPPFCVARCPSGVKPDLSYMPIWKFPDEEGACQPCPINC THSCVDLDDKGCPAEQRASPLTSIISAVVGILLVVVLGVVFGILIKRRQQ KIRKYTMRRLLQETELVEPLTPSGAMPNQAQMRILKETELRKVKVLGSGA FGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSP YVSRLLGICLTSTVQLVTQLMPYGCLLDHVRENRGRLGSQDLLNWCMQIA KGMSYLEDVRLVHRDLAARNVLVKSPNHVKITDFGLARLLDIDETEYHAD GGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPARE IPDLLEKGERLPQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMA RDPQRFVVIQNEDLGPASPLDSTFYRSLLEDDDMGDLVDAEEYLVPQQGF FCPDPAPGAGGMVHHRHRSSSTRSGGGDLTLGLEPSEEEAPRSPLAPSEG AGSDVFDGDLGMGAAKGLQSLPTHDPSPLQRYSEDPTVPLPSETDGYVAP LTCSPQPEYVNQPDVRPQPPSPREGPLPAARPAGATLERPKTLSPGKNGV VKDVFAFGGAVENPEYLTPQGGAAPQPHPPPAFSPAFDNLYYWDQDPPER GAPPSTFKGTPTAENPEYLGLDVPV
Attempts have been made to utilize E75 as an anti-cancer vaccine, for example, as a single peptide vaccine combined with different immunoadjuvants in patients with advanced cancer who overexpress the HER2/neu protein (Zaks T Z et al. (1998) Cancer Res. 58:4902-8; Knutson K L et al. (2002) Clin. Cancer Res. 8:1014-8; and, Murray J L et al. (2002) Clin. Cancer Res. 8:3407-18); loaded on to autologous dendritic cells and reinfused (Brossart P et al. (2000) Blood 96:3102-8; and, Kono K et al. (2002) Clin. Cancer Res. 8:3394-3400); or embedded in longer peptides capable of binding HLA class II molecules in order to recruit CD4 helper T-cells (Disis M L et al. (1999) Clin. Cancer Res. 5:1289-97; and, Disis M L et al. (2002) J. Clin. Oncol. 20:2624-32). Each approach has stimulated an E75-specific cytotoxic T cell-mediated immune response, but none has demonstrated a clinically significant therapeutic or protective immunity in women with advanced stage breast cancer. The inability of others to show a meaningful clinical benefit using E75 vaccine preparations stems in part from the fact that E75 is derived from a “self” tumor antigen. Cancer vaccines targeting “self” tumor antigens, like those derived from HER2/neu, present unique challenges because of the immunologic tolerance characteristic of self proteins. Furthermore, the previous studies have focused on cancer patients with advanced disease, such as Stage III or IV cancer, rather than patients who are disease-free following standard therapies. As such, none of these attempts to use E75 as an anti-cancer vaccine has demonstrated the vaccine's ability to prevent or delay recurrence of disease following remission. Building on these E75 studies, others have more recently conducted clinical trials to determine if E75-induced immunity conveys a clinical benefit by preventing the recurrence in high-risk breast cancer patients. Peoples G E et al., J. Clin. Oncol. (2005) 23:7536-45; Peoples G E et al., Clin Cancer Res (2008) 14(3):797-803; Holmes et al., Cancer (2008) 113:1666-75. The data from these studies indicate that increased in vivo E75-induced DTH responses correlate with reduced recurrence and increased survival time for those who did recur.
GP2, initially described by Peoples et al., is a nine amino acid peptide derived from the transmembrane portion of the HER2/neu protein corresponding to amino acids 654-662 of the full length sequence (i.e., IISAVVGIL: SEQ ID NO:2) (Peoples G E et al., Proc Natl Acad Sci USA (1995) 92:432-436, which is hereby incorporated by reference in its entirety). The peptide was isolated using tumor-associated lymphocytes from patients with breast and ovarian cancer, and later found to be shared amongst several epithelial malignancies including non-small cell lung cancer and pancreatic cancer (Linehan D C et al., J Immunol (1995) 155:4486-4491; Peiper M et al., Surgery (1997) 122:235-242; Yoshino I et al., Cancer Res (1994) 54:3387-3390; Peiper M et al., Eur J Immunol (1997) 27:1115-1123).
E75 has a high binding affinity for the HLA-A2 molecule and is considered the immunodominant peptide of the HER2/neu protein. As such it is the most studied HER2/neu-derived peptide in laboratory and clinical studies. Peoples et al., J. Clin. Oncol. (2005) 23:7536-45. As the immunodominant peptide, E75 is also expected to induce a more potent immune response. GP2, on the other hand, has a relatively poor binding affinity to HLA-A2 and is considered a subdominant epitope. (Fisk B, et al. J Exp Med (1995) 181:2109-2117.) This is one of the reasons that vaccine strategies targeting a cellular immune response to HER-2/neu epitopes have focused on E75 rather than GP2.
Previous studies of GP2 have used autologous dendritic cells pulsed with GP2 (and other peptides) ex vivo and re-injected subcutaneously (Brossart P et al. Blood (2000) 96:3102-3108) or intravenously (Dees E C et al. Cancer Immunol Immunother (2004) 53:777-785) into HER2/neu+ patients with metastatic breast or ovarian cancer to induce a CTL response. Brossart et al. detected a peptide-specific (GP2 and E75) CTL response in vivo, and they noted that both peptides showed a similar immune response despite known differences in HLA-A2 binding affinities. Dees et al. evaluated GP2-pulsed dendritic cells in metastatic breast cancer patients and were able to document clinically stable disease in two patients. Importantly, however, neither study used GP2 as a peptide vaccine. Rather in both studies, patients were injected with dendritic cells that had been pulsed with GP2. Furthermore, as with the E75 studies, the GP2 studies were limited to patients with advanced cancer. Therefore, neither Brossart nor Dees demonstrated the ability of GP2-pulsed dendritic cells to prevent or delay recurrence of disease following remission. As with E75, cancer vaccines targeting “self” tumor antigens, like HER2/neu from which GP2 is derived, present unique challenges because of the immunologic tolerance characteristic of self proteins.
Peoples et al. have previously evaluated the use of GP2 for a peptide-based breast cancer vaccine trial by conducting in vitro cytotoxicity assays with GP2-pulsed dendritic cells and CD8 T cells obtained from breast cancer patients. (Mittendorf E A et al. Cancer (2006) 106:2309-2317.) While the results from these in vitro experiments confirmed the presence of GP2-specific precursor cytotoxic T lymphocytes in women with HER2/neu+ breast cancer, it was concluded that because of the variability of response to a given peptide and the heterogeneity of antigen expression in vivo, vaccination with multiple different peptides, including the immunodominant peptide E75, will be required to provide an adequate immune response. (Mittendorf E A et al. Cancer (2006) 106:2309-2317.)
As noted above, trastuzumab is indicated for HER2/neu over-expressing (IHC 3+ or FISH≧2.0), node-positive (NP), metastatic breast cancer patients, and shows very limited activity in patients with low to intermediate HER2/neu expression. Similarly, in the studies discussed above, patients receiving the E75 and GP2-based vaccines were selected, in part, based on the presence of tumors that over-expressed HER2/neu. Accordingly, a GP2 peptide vaccine would not be expected to be effective in cancer patients with low and intermediate levels of HER2/neu tumor expression.